Ligation and/or assembly of nucleic acid molecules

Abstract

The present invention relates to ligation and/or assembly of nucleic acid molecules. Particularly, a double-stranded target nucleic acid having overhangs of at least one nucleotide is ligated with another nucleic acid molecule capable of forming a stem-loop structure with an overhang of at least one nucleotide. The invention is suitable for tagging nucleic acid molecules. In specific embodiments, the overhangs can be produced by chemical cleavage of phosphorothioate-modified nucleic acid molecules. The invention further relates to the amplification of the ligated product, and using the resultant amplicon for assembly of multiple nucleic acid fragments.

The invention claimed is: 
     
       1. A method for ligating three nucleic acid molecules comprising:
 (i) providing a first nucleic acid molecule comprising a first overhang of one nucleotide in length at a first end and a second overhang of one nucleotide of in length at its other (or second) end; wherein the first overhang and the second overhang are different nucleotides and are not complementary to each other, wherein the first overhang is a 3′ C overhang and the second overhang is a 3′ T overhang, wherein the first nucleic acid molecule is a double-stranded nucleic acid molecule comprising a coding sequence strand and a substantially complementary strand, and wherein the 3′ C overhang is on the substantially complementary strand and the 3′ T overhang is on the coding sequence strand; 
 (ii) providing a second nucleic acid molecule capable of forming a stem-loop structure with an overhang of one nucleotide; wherein the overhang of the second nucleic acid molecule is substantially complementary to the first overhang of the first end of the first nucleic acid molecule, wherein the overhang of the second nucleic acid molecule is a 3′ G overhang; and also providing a third nucleic acid molecule capable of forming a stem-loop structure with an overhang of one nucleotide; wherein the overhang of the third nucleic acid molecule is substantially complementary to the second overhang of the second end of the first nucleic acid molecule, wherein the overhang of the third nucleic acid molecule is a 3′ A overhang; and wherein the overhang of the second nucleic acid molecule and the overhang of the third nucleic acid molecule are different nucleotides and are not complementary to each other; and
 (iii) ligating the first overhang at the first end of the first nucleic acid molecule to the overhang of the second nucleic acid molecule and also the second overhang of the second end of the first nucleic acid molecule to the overhang of the third nucleic acid molecule to form a single nucleic acid molecule. 
 
 
     
     
       2. The method according to  claim 1 ; wherein the second nucleic acid comprises a first defined sequence and the third nucleic acid comprises a second defined sequence. 
     
     
       3. The method according to  claim 2 ; wherein the first defined sequence of the second nucleic acid molecule comprises a first tag sequence, a first barcode sequence and/or a first linking sequence and the second defined sequence of the third nucleic acid molecule comprises a second tag sequence; a second barcode sequence and/or a second linking sequence. 
     
     
       4. The method according to  claim 1 ; wherein step (i) comprises:
 (i)(a) providing a double-stranded nucleic acid template comprising a first nucleic acid strand and a second nucleic acid strand substantially reverse complementary to the first nucleic acid strand; 
 (i)(b) providing a first primer comprising a first sequence with at least one modified nucleotide upstream of a second sequence substantially complementary to the second strand of the nucleic acid template; and a second primer comprising a third sequence with at least one modified nucleotide upstream of a fourth sequence substantially complementary to the first strand of the nucleic acid template; 
 (i)(c) amplifying the nucleic acid template using the first and second primers to produce an amplicon; and 
 (i)(d) chemically cleaving the amplicon to produce the first nucleic acid molecule comprising a first overhang of one nucleotide in length at a first end and a second overhang of at least one nucleotide of one nucleotide in length at its other (or second) end; wherein the first overhang and the second overhang are different nucleotides and are not complementary to each other; or 
 (i)(a) providing a first single-stranded nucleic acid molecule; 
 (i)(b) providing a second single-stranded nucleic acid molecule substantially complementary to the first single nucleic acid molecule; and 
 (i)(c) allowing the first and second single-stranded nucleic acid molecule to anneal to produce the first nucleic acid molecule comprising a first overhang of one nucleotide in length at a first end and a second overhang of one nucleotide in length at its other (or second) end; wherein the first overhang and the second overhang are different nucleotides and are not complementary to each other. 
 
     
     
       5. The method according to  claim 1 , further comprising the steps of:
 (iv) using the single nucleic acid molecule as a template from step (iii) for amplifying in a polymerase chain reaction with two amplification primers to produce an amplicon;
 (v) performing a ligation to join the amplicon to a plurality of nucleic acid molecules to form an assembly of joined plurality of nucleic acid molecules. 
 
 
     
     
       6. The method according to  claim 5 ; wherein step (iv) comprises amplifying the template with a first amplification primer comprising at least one modified nucleotide and a second amplification primer comprising at least one modified nucleotide; further chemically cleaving the amplicon to produce a first end with a third overhang and a second end with a fourth overhang. 
     
     
       7. The method according to  claim 5 ; wherein each of the plurality of nucleic acid molecules is an amplicon from step (iv) using another single nucleic acid molecule from step (iii) as a template. 
     
     
       8. The method according to  claim 5 , wherein the amplicon and each of the plurality of nucleic acid molecules have different sequences. 
     
     
       9. The method according to  claim 5 , wherein step (v) comprises ligating to form a concatemer of nucleic acid molecules, each with substantially the same sequence. 
     
     
       10. The method according to  claim 5 , wherein the assembly of joined plurality of nucleic acid molecules is circular. 
     
     
       11. The method according to  claim 2 , further comprising the steps of:
 (iv) using the single nucleic acid molecule from step (iii) as a template for amplifying in a polymerase chain reaction with an amplification primer having a sequence designed based on at least part or all of the first defined sequence and comprising at least one modified nucleotide and another amplification primer having a sequence designed based on at least part or all of the second defined sequence and comprising at least one modified nucleotide to produce an amplicon, chemically cleaving the amplicon to produce a first end with a third overhang and a second end with a fourth overhang; and 
 (v) performing a ligation to join the amplicon to a plurality of nucleic acid molecules to form an assembly of joined plurality of nucleic acid molecules; wherein each of the plurality of nucleic acid molecules is an amplicon from step (iv). 
 
     
     
       12. The method according to  claim 11 , wherein each of the plurality of nucleic acid molecules is an amplicon from step (iv) using another single nucleic acid molecule from step (iii) as a template. 
     
     
       13. The method according to  claim 11 , wherein amplification primers designed based on defined sequences of a said second nucleic acid molecules comprising a stem-loop structure as applicable are used to order and/or arrange the plurality of nucleic acid molecules in the assembly. 
     
     
       14. The method according to  claim 11 , wherein the amplicon and each of the plurality of nucleic acid molecules have different sequences. 
     
     
       15. The method according to  claim 11 , wherein the assembly of joined plurality of nucleic acid molecules is circular. 
     
     
       16. The method according to  claim 15 , wherein the method further comprises using polymerase chain reaction with amplification primers designed based on applicable defined sequences to implement a modification in the assembly of joined plurality of nucleic acid molecules, wherein the modification comprises inserting at least one nucleic acid molecule into the assembly, removing at least one joined nucleic acid molecule from the assembly or replacing at least one joined nucleic acid molecule in the assembly. 
     
     
       17. The method according to  claim 16 , comprising implementing the modification to form a library of different plasmids.